Virulence gene expression in many pathogenic bacteria is controlled by environmental stimuli both inside and outside of the host. For the human pathogen Vibrio cholerae, the coordinate expression of cholera toxin (CT), toxin-coregulated pilus (TCP), and a number of other virulence factors is strongly influenced by environmental parameters such as temperature, pH, osmolarity, and oxygen tension. To elucidate the nature of this regulation, a transcriptional fusion between the promoter of the CT operon (ctx) and the Escherichia coli b-galactosidase (lacZ) gene in the V. cholerae chromosome was used as a screen to identify genes that influence the expression of the fusion in response to various environmental stimuli. Using this screen, two V. cholerae genes have been identified, cya and crp, that negatively regulate virulence gene expression under particular growth conditions. The product of the cya gene, adenylate cyclase, catalyzes the synthesis of cyclic AMP (cAMP), one of the most important and ubiquitous intracellular regulatory molecules. Together with the crp product, cAMP-receptor protein (CRP), this system serves as a global regulator of gene expression in enteric bacteria by controlling the utilization of carbon and energy sources in the environment. The observation that crp mutants in both the classical and El Tor biotypes of V. cholerae O1 are derepressed for the expression of CT and TCP under a variety of growth conditions in vitro and are defective for pathogenesis in the infant mouse cholera model is the first clear demonstration that cAMP-CRP plays a role in these processes. The primary focus of the proposed work is to elucidate the molecular and physiological mechanisms by which cAMP-CRP controls the expression of CT and TCP in V. cholerae. This will be accomplished in SPECIFIC AIM #1 by determining which regulatory genes are influenced by cAMP-CRP and by identifying the site(s) to which CRP binds, and in SPECIFIC AIM #2 by examining the relationship between the intracellular levels of cAMP under various growth conditions and the expression of CT and TCP. Since analysis of the crp mutants suggests that in addition to cAMP-CRP, other as yet unknown factors also influence the environmental control of virulence gene expression, the third SPECIFIC AIM of this proposal is to again exploit the ctx-lacZ fusion screen to identify these genes in both biotype strains and to shed light on their mechanisms of action. The long-term objectives of this work are to ultimately understand how stimuli from the environment are transmitted to the level of gene expression in V. cholerae so as to facilitate the development of new strategies to control and prevent bacterial diseases.